1. Molecular Cloning: Construction of a recombinant DNA

2. Molecular Cloning: Cloning Vectors
Plasmids

3. Grand-daddy of plasmids

4. Molecular Cloning: Cloning Vectors
Plasmids
Origin of replication, determines the number of copies per cell
Marker genes: ampicillin and tetracycline resistance genes
Unique restriction enzyme cut sites

5. Creation of a polylinker or multiple cloning site

6. Molecular Cloning: Cloning Vectors
1. Plasmids
Origin of replication, determines the number of copies per cell
Marker genes: ampicillin and tetracycline resistance genes
Unique restriction enzyme cut sites
Polylinker of MCS
Small size
Limitation is ~15,000 bp

7. Entry of DNA into cells Chemical Transformation Electroporation
Treat cells with calcium chloride
Heat shock
Electroporation
High voltage transiently makes bacterial membrane permeable

8. Special examples of different plasmids
Expression vectors

10. Special examples of different plasmids
A. Expression vectors
Strong promoter for transcription
Ribosome binding site
Transcription terminator
Some way of controlling gene – making it inducible

11. Tag expressed protein with another protein or short peptide
Fusion proteins
Tag expressed protein with another protein or short peptide

12. Type of Tags Fluorescent proteins
Examples is the green fluoresecent protein or GFP

14. Type of Tags Fluorescent proteins Epitope tag
Examples is the green fluoresecent protein or GFP
Epitope tag
Short peptide sequence which has an antibody that recognizes it specifically

16. Type of Tags Fluorescent proteins Epitope tag Metal chelator
Examples is the green fluoresecent protein or GFP
Epitope tag
Short peptide sequence which has an antibody that recognizes it specifically
Metal chelator
Can bind to Ni or Co chelated and immobolized

17. Special examples of different plasmids
Shuttle vectors
Contains origins of replicatioin from two different organisms
Can be replicated in both
Often used to shuttle plasmids from bacteria to
Cosmids – related to bacteriophages

18. Molecular Cloning: Cloning Vectors
2. Bacteriophages: example is lambda
1/3 of genome (48.5 Kb) is non essential
DNA is packaged into phage particles
Can only fit 40 – 53 Kb of DNA
Have an in vitro packaging system
Highly efficient at transforming bacteria
Can clone up to 23 Kb of DNA

20. Molecular Cloning: Cloning Vectors
3. Bacterial Artificial Chromosomes (BAC)
100 to 300 Kb in size
Have selectable markers
Stable origin of replication
Size of inserts is ~100 KB
Uses electroporation

22. Molecular Cloning: Cloning Vectors
4. Yeast Artificial Chromosomes

24. Molecular Cloning: Cloning Vectors
4. Yeast Artificial Chromosomes (YAC)
ARS or origin of replication
Selectable markers
CEN or centromere sequence for proper segregation
Telomere sequences
Suitable for very large DNAs

25. A DNA library Collection of DNA clones Source for gene discovery
Largest example is genomic library
Other subsets would be such things as:
cDNA libraries
Can scan these libraries by DNA hybridization